EP1123304B1 - Flavonoids derived from citrus peels as collagen-induced platelet aggregation inhibitor - Google Patents

Flavonoids derived from citrus peels as collagen-induced platelet aggregation inhibitor Download PDF

Info

Publication number
EP1123304B1
EP1123304B1 EP99938643A EP99938643A EP1123304B1 EP 1123304 B1 EP1123304 B1 EP 1123304B1 EP 99938643 A EP99938643 A EP 99938643A EP 99938643 A EP99938643 A EP 99938643A EP 1123304 B1 EP1123304 B1 EP 1123304B1
Authority
EP
European Patent Office
Prior art keywords
platelet aggregation
collagen
induced platelet
flavonoid
citrus peels
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP99938643A
Other languages
German (de)
French (fr)
Other versions
EP1123304A1 (en
Inventor
Song Hae Bok
Tae Sook Jeong
Han Ik Cho
Dong Soon Lee
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Korea Research Institute of Bioscience and Biotechnology KRIBB
Original Assignee
Korea Research Institute of Bioscience and Biotechnology KRIBB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Korea Research Institute of Bioscience and Biotechnology KRIBB filed Critical Korea Research Institute of Bioscience and Biotechnology KRIBB
Publication of EP1123304A1 publication Critical patent/EP1123304A1/en
Application granted granted Critical
Publication of EP1123304B1 publication Critical patent/EP1123304B1/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/75—Rutaceae (Rue family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/752—Citrus, e.g. lime, orange or lemon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to a use of flavonoids derived from citrus peels for inhibiting a collagen-induced platelet aggregation in a mammal.
  • the flavonoids can be administered to the mammal in the form of a pharmaceutical composition or a food composition.
  • US-A-4842859 describes a pharmaceutical composition comprising plant extracts in which flavonoids named Aurapten and Scoparone reduce platelet aggregation. These flavonoids are extracted from citrus peels.
  • Platelet aggregation on a blood vessel is induced by several factors, e.g., collagen, adenosine diphosphate (ADP), thrombin, epinephrine, ristocetin and arachidonic acid.
  • ADP adenosine diphosphate
  • thrombin adenosine diphosphate
  • epinephrine adenosine diphosphate
  • arachidonic acid e.g., adenosine diphosphate (ADP), thrombin, epinephrine, ristocetin and arachidonic acid.
  • ADP adenosine diphosphate
  • thrombin epinephrine
  • ristocetin arachidonic acid
  • thromboxane A2 inhibitor PGG/H synthase inhibitor, thromoxane synthase inhibitor, thromboxane A2 receptor antagonist, ticlopidine, GP Hb-IHa inhibitor and serotonin antagonist
  • drugs e.g., thromboxane A2 inhibitor, PGG/H synthase inhibitor, thromoxane synthase inhibitor, thromboxane A2 receptor antagonist, ticlopidine, GP Hb-IHa inhibitor and serotonin antagonist.
  • Ticlopidine and analogues thereof selectively inhibit the ADP-induced platelet aggregation in vivo and are known to be effective in the treatment of a transient ischemic cerebral stroke and peripheral arterial or ischemic heart disease(Hass, W. K. et al., N. Engl. J. Med. , 21 , 501(1989); and Easton, J. D., Drugs , 42 , 39(1991)).
  • these drugs have been reported to have such undesirable side effects as myelosuppression, increase of total cholesterol level, diarrhea, eruption and neutropenia((McTavish, E.
  • the flavonoids extracted from citrus peel e.g., naringin, naringenin, hesperidin and hesperetin
  • flavonoids extracted from citrus peel e.g., naringin, naringenin, hesperidin and hesperetin
  • both hesperidin and hesperetin have capillary-enhancing, permeability-reducing, anti-inflammation, antiviral, and blood pressure- and cholesterol-lowering activities(Meyer, O. C., Angiology , 45 , 579-584(1994); Struckmann, J. R. et al., Angiol. , 45 , 419-428(1994); Matsubara, Y.
  • naringin and naringenin have anticancer, anti-ulcer, and cholesterol-lowering activities(Monforte, M. T. et al., Farmaco. , 50 (9), 595-599(1995, Sep.); JP 95-86929; JP 95-86930; Felicia, V. et al., Nutr. Cancer , 26 , 167-181(1996); EP 0352147 A2 (1990.1.24); and Martin, M. J. et al., Parmacol. , 49 , 144-150 (1994)).
  • naringenin inhibits an arachidonic acid-induced platelet aggregation (Corcazier, E. et al., Biochimica Biophysica Acta , 835 , 315-321(1985)).
  • a flavonoid derived from citrus peels for inhibiting a collagen-induced platelet aggregation.
  • flavonoids selected from the group consisting of naringin, naringenin, hesperetin and a mixture thereof for the manufacture of a medicament can be administered to a mammal in the form of a pharmaceutical composition or a food composition.
  • flavonoids may also be synthesized according to the processes described by Zemplen, Bognar, Ber. , 75 , 1043(1943) and Seka, Prosche, Monatsh. , 69 , 284(1936). Further, naringenin and hesperetin can be prepared by the hydrolysis of naringin and hesperidin, respectively.
  • the flavonoids exert a selective inhibitory effect on a collagen-induced platelet aggregation as well as thrombogenesis.
  • the flavonoids show little toxicity or mitogenicity in animal tests. More specifically, the flavonoids exhibit no toxicity when they are orally administered to a mouse at a dosage of 1,000 mg/kg, which corresponds to an oral administration dose of 3 to 10 g per kg of body weight. Further, the flavonoids exert no adverse effects on the liver function.
  • the present invention also provides a pharmaceutical composition for inhibiting a collagen-induced platelet aggregation, which comprises an effective amount of a flavonoid derived from citrus peels as an active ingredient together with pharmaceutically acceptable excipients, carriers or diluents.
  • the pharmaceutical formulation may be prepared by using any of the conventional procedures.
  • the active ingredient is preferably admixed or diluted with a carrier, or enclosed within a carrier which may be in the form of a capsule, sachet or other container.
  • the carrier serves as a diluent, it may be a solid, semi-solid or liquid material acting as a vehicle, excipient or medium for the active ingredient.
  • the formulation may be in the form of a tablet, pill, powder, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, soft and hard gelatin capsule, sterile injectable solution, sterile packaged powder and the like.
  • Suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate and mineral oil.
  • the formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like.
  • the compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a mammal by employing any of the procedures well known in the art.
  • the pharmaceutical formulation of the present invention can be administered via various routes including oral, transdermal, subcutaneous, intravenous and intramuscular introduction.
  • a typical daily dose of flavonoid may range from about 0.5 to 100 mg/kg body weight, preferably 2 to 10 mg/kg body weight, and can be administered in a single dose or in divided doses.
  • the amount of the active ingredient actually administered ought to be determined in light of various relevant factors including the condition to be treated, the chosen route of administration, the age, sex and body weight of the individual patient, and the severity of the patient's symptom; and, therefore, the above dose should not be intended to limit the scope of the invention in any way.
  • the flavonoid can be incorporated in foods, as an additive or dietary supplement, for the purpose of inhibiting the collagen-induced platelet aggregation.
  • the present invention also provide a food composition for inhibiting the collagen-induced platelet aggregation, which comprises an effective amount of the flavonoid.
  • the foods may include meats; chocolates; snacks; confectionery; pizza; foods made from cereal flour such as breads, cakes, crackers, cookies, biscuits, noodles and the likes; gums; dairy products such as milk, cheese, yogurt and ice creams; soups; broths; pastes, ketchups and sauces; vitamin complex; and various health foods.
  • the content of the flavonoid in a food may range from 0.1 to 50% by weight.
  • the flavonoid can be used as an effective, non-toxic pharmaceutical agent for inhibiting the collagen-induced platelet aggregation.
  • Example 1 Activity of Flavonoid in Platelet Aggregation Inhibition
  • Inhibitory activity of the citrus peel flavonoids on the platelet aggregation was determined as follows, in accordance with the methods of O'Brein(O'Brein JR., J. Clin. Pathol. , 15 , 452(1962)) and Aok(Aok, H. et al., anesthesiology , 88 , 362(1998)).
  • a platelet aggrometer (Platelet aggrecoder II, PA-3220, Chronolog, U.S.A.). Then, 50 ul of aggregation enhancer solution, i.e., 2 mg/ml collagen solution, 100 uM ADP, 200 uM epinephrine or 15 mg/ml ristocetin, was added thereto. The resulting mixture was reacted at 37 °C for 3 min. and then the optical density thereof was determined. To determine the base line, platelet-free plasma was used.
  • naringin, naringenin, hesperidin and hesperetin exhibited an inhibitory activity on the collagen-induced platelet aggregation while they do not inhibit any of ADP-, epinephrine- and ristocetin-induced platelet aggregation.
  • Naringin, naringenin, hesperidin and hesperetin inhibited the collagen-induced platelet aggregation by 59 to 85 %, 30 to 87 %, 59 to 93 %, and 15 to 93 %, respectively.
  • the result shows that the flavonoid derived from citrus peels selectively inhibit the collagen-induced platelet aggregation.
  • mice(8 heads) each weighing about 25 to 29 g and male mice(8 heads) each weighing about 34 to 38 g were bred under the condition of temperature 22 ⁇ 1°C, moisture 55 ⁇ 5% and photoperiod 12L/12D.
  • Fodder(Cheiljedang Co., mouse and rat fodder) and water were sterilized and fed to the mice.
  • a flavonoid derived from citrus peel, naringin or hesperidin was dissolved in 0.5% Tween 80 to a concentration of 100 mg/ml, and the solution was orally administered to the mice in an amount of 0.2 ml per 20 g of mouse body weight.
  • the solution was administered once and the mice were observed for 10 days for signs of adverse effects or death according to the following schedule: 1, 4, 8, and 12 hours after the administration and, every 12 hours thereafter.
  • the weight changes of the mice were recorded every day to examine the effect of the flavonoid derived from citrus peels. Further, on the 10th day, the mice were sacrificed and the internal organs were visually examined.
  • mice were alive at day 10 and, the flavonoid derived from citrus peels showed no toxicity at a dose of 1,000 mg/kg.
  • the autopsy revealed that the mice did not develop any pathological abnormality, and no weight loss was observed during the 10 day test period. Conclusively, the flavonoid derived citrus peels is not toxic when orally administered to an animal.
  • Example 3 Pharmaceutical Formulation Containing Flavonoid Derived From Citrus Peels
  • Hard gelatin capsules were prepared using the following ingredients: Quantity (mg/capsule) Active ingredient (Naringin, naringenin, hesperidin, or hesperetin) 200 Vitamin C 100 Starch, dried 180 Magnesium stearate 20 Total 500 mg
  • Foods containing a flavonoid derived from citrus peels were prepared as follows.
  • naringin, naringenin, hesperidin or hesperetin was added to the coagulated milk protein; and, in case of yogurt preparation, naringin, naringenin, hesperidin or hesperetin was added to the coagulated milk protein obtained after the fermentation.

Abstract

The present invention relates to a use of a flavonoid derived from citrus peels for inhibiting a collagen-induced platelet aggregation in a mammal.

Description

FIELD OF THE INVENTION
The present invention relates to a use of flavonoids derived from citrus peels for inhibiting a collagen-induced platelet aggregation in a mammal. The flavonoids can be administered to the mammal in the form of a pharmaceutical composition or a food composition.
BACKGROUND OF THE INVENTION
US-A-4842859 describes a pharmaceutical composition comprising plant extracts in which flavonoids named Aurapten and Scoparone reduce platelet aggregation. These flavonoids are extracted from citrus peels.
The document "An. R. Acad. Farm. (1986), 52(3), pages 497-504)" refers to the use of hesperidin for the purpose of inhibiting platelet aggregation. The flavonoids used therein are extracted from citrus peels.
Platelet aggregation on a blood vessel is induced by several factors, e.g., collagen, adenosine diphosphate (ADP), thrombin, epinephrine, ristocetin and arachidonic acid. For instance, when epithelial cells of a blood vessel are damaged, collagen underneath the epithelial cells is exposed to blood stream and activates platelets. The activation of platelets triggers a cascade of reactions resulting in a thrombogenesis and platelet aggregation on the blood vessel, which may cause serious circulatory diseases, e.g., thrombosis, atherosclerosis, cerebral stroke and coronary artery thromboembolism.
Hitherto, various drugs, e.g., thromboxane A2 inhibitor, PGG/H synthase inhibitor, thromoxane synthase inhibitor, thromboxane A2 receptor antagonist, ticlopidine, GP Hb-IHa inhibitor and serotonin antagonist, have been developed in order to prevent such platelet aggregation. However, these drugs have shown various undesirable side effects on the internal organs, and hemorrhagenic properties.
Ticlopidine and analogues thereof, e.g., clopidogrel, selectively inhibit the ADP-induced platelet aggregation in vivo and are known to be effective in the treatment of a transient ischemic cerebral stroke and peripheral arterial or ischemic heart disease(Hass, W. K. et al., N. Engl. J. Med., 21, 501(1989); and Easton, J. D., Drugs, 42, 39(1991)). However, these drugs have been reported to have such undesirable side effects as myelosuppression, increase of total cholesterol level, diarrhea, eruption and neutropenia((McTavish, E. et al., 2Drugs, 40, 238(1990)); Hass, W. K. et al., supra; and Dunn C. D. R., Scrip. Reports, Stroke: Trends, Treatments and Markets, PJB Publications Ltd. pp133-139(1995)).
Accordingly, there has continued to exist a need to develop a platelet aggregation inhibitor having a highly selective inhibitory activity on the collagen-induced platelet aggregation and yet without incurring adverse side effects.
It is known that. the flavonoids extracted from citrus peel, e.g., naringin, naringenin, hesperidin and hesperetin, have activities in improving lipid metabolism to prevent cardio-circulatory diseases, as well as anticancer and antiviral activities. In particular, it has been reported that both hesperidin and hesperetin have capillary-enhancing, permeability-reducing, anti-inflammation, antiviral, and blood pressure- and cholesterol-lowering activities(Meyer, O. C., Angiology, 45, 579-584(1994); Struckmann, J. R. et al., Angiol., 45, 419-428(1994); Matsubara, Y. et al., Japan Organic Synthesis Chem. Association Journal, 52, 318-327(1994. Mar.); Galati, E. M. et al., Farmaco., 51(3), 219-221(1996, Mar.); Monforte, M. T. et al., Farmaco., 50(9), 595-599(1995, Sep.); JP 95-86929; JP 95-86930; Galati, E. M. et al., Farmaco., 40(11), 709-712(1994, Nov.); and Emim, J. A. et al., J. Pharm. Pharmacol., 46(2), 118-122(1994)). Further, it has been reported that both naringin and naringenin have anticancer, anti-ulcer, and cholesterol-lowering activities(Monforte, M. T. et al., Farmaco., 50(9), 595-599(1995, Sep.); JP 95-86929; JP 95-86930; Felicia, V. et al., Nutr. Cancer, 26, 167-181(1996); EP 0352147 A2 (1990.1.24); and Martin, M. J. et al., Parmacol., 49, 144-150 (1994)).
Recently, it has also been reported that naringenin inhibits an arachidonic acid-induced platelet aggregation (Corcazier, E. et al., Biochimica Biophysica Acta, 835, 315-321(1985)).
However, there has been no report on the selective inhibitory activity of flavonoids derived from citrus peels on a collagen-induced platelet aggregation.
SUMMARY OF THE INVENTION
Accordingly, it is a primary object of the present invention to provide an inhibitor of a collagen-induced platelet aggregation.
In accordance with the present invention, there is provided a use of a flavonoid derived from citrus peels for inhibiting a collagen-induced platelet aggregation.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, flavonoids selected from the group consisting of naringin, naringenin, hesperetin and a mixture thereof for the manufacture of a medicament can be administered to a mammal in the form of a pharmaceutical composition or a food composition.
These flavonoids may also be synthesized according to the processes described by Zemplen, Bognar, Ber., 75, 1043(1943) and Seka, Prosche, Monatsh., 69, 284(1936). Further, naringenin and hesperetin can be prepared by the hydrolysis of naringin and hesperidin, respectively.
The flavonoids exert a selective inhibitory effect on a collagen-induced platelet aggregation as well as thrombogenesis.
Moreover, in spite of their potent efficacy, the flavonoids show little toxicity or mitogenicity in animal tests. More specifically, the flavonoids exhibit no toxicity when they are orally administered to a mouse at a dosage of 1,000 mg/kg, which corresponds to an oral administration dose of 3 to 10 g per kg of body weight. Further, the flavonoids exert no adverse effects on the liver function.
The present invention also provides a pharmaceutical composition for inhibiting a collagen-induced platelet aggregation, which comprises an effective amount of a flavonoid derived from citrus peels as an active ingredient together with pharmaceutically acceptable excipients, carriers or diluents.
The pharmaceutical formulation may be prepared by using any of the conventional procedures. In preparing the formulation, the active ingredient is preferably admixed or diluted with a carrier, or enclosed within a carrier which may be in the form of a capsule, sachet or other container. When the carrier serves as a diluent, it may be a solid, semi-solid or liquid material acting as a vehicle, excipient or medium for the active ingredient. Thus, the formulation may be in the form of a tablet, pill, powder, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, soft and hard gelatin capsule, sterile injectable solution, sterile packaged powder and the like.
Examples of suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a mammal by employing any of the procedures well known in the art.
The pharmaceutical formulation of the present invention can be administered via various routes including oral, transdermal, subcutaneous, intravenous and intramuscular introduction. In case of human, a typical daily dose of flavonoid may range from about 0.5 to 100 mg/kg body weight, preferably 2 to 10 mg/kg body weight, and can be administered in a single dose or in divided doses. However, it should be understood that the amount of the active ingredient actually administered ought to be determined in light of various relevant factors including the condition to be treated, the chosen route of administration, the age, sex and body weight of the individual patient, and the severity of the patient's symptom; and, therefore, the above dose should not be intended to limit the scope of the invention in any way.
Moreover, the flavonoid can be incorporated in foods, as an additive or dietary supplement, for the purpose of inhibiting the collagen-induced platelet aggregation. Accordingly, the present invention also provide a food composition for inhibiting the collagen-induced platelet aggregation, which comprises an effective amount of the flavonoid. The foods may include meats; chocolates; snacks; confectionery; pizza; foods made from cereal flour such as breads, cakes, crackers, cookies, biscuits, noodles and the likes; gums; dairy products such as milk, cheese, yogurt and ice creams; soups; broths; pastes, ketchups and sauces; vitamin complex; and various health foods. In this case, the content of the flavonoid in a food may range from 0.1 to 50% by weight.
As described above, the flavonoid can be used as an effective, non-toxic pharmaceutical agent for inhibiting the collagen-induced platelet aggregation.
The following Examples are intended to further illustrate the present invention without limiting its scope.
Further, percentages given below for solid in solid mixture, liquid in liquid, and solid in liquid are on a wt/wt, vol/vol and wt/vol basis, respectively, and all the reactions were carried out at room temperature, unless specifically indicated otherwise.
Example 1: Activity of Flavonoid in Platelet Aggregation Inhibition
Inhibitory activity of the citrus peel flavonoids on the platelet aggregation was determined as follows, in accordance with the methods of O'Brein(O'Brein JR., J. Clin. Pathol., 15, 452(1962)) and Aok(Aok, H. et al., anesthesiology, 88, 362(1998)).
9 ml of human blood was added to 1 ml of 3.8 % sodium citrate and the mixture was centrifuged at 1,000 rpm for 10 min. The supernatant(plasma) was diluted with phosphate buffered saline(PBS) to a concentration of 200,000 to 400,000 platelets/mm3. Each 450 ul of the diluted plasma was put in the cuvettes and 5 ul of a flavonoid solution, wherein naringin, naringenin, hesperidin or hesperetin is dissolved in dimethylsulfoxide at a concentration of 10 mg/L, was added thereto, except for one control cuvette. The mixture was warmed at 37 °C for 3 min. in a platelet aggrometer(Platelet aggrecoder II, PA-3220, Chronolog, U.S.A.). Then, 50 ul of aggregation enhancer solution, i.e., 2 mg/ml collagen solution, 100 uM ADP, 200 uM epinephrine or 15 mg/ml ristocetin, was added thereto. The resulting mixture was reacted at 37 °C for 3 min. and then the optical density thereof was determined. To determine the base line, platelet-free plasma was used.
Consequently, all of naringin, naringenin, hesperidin and hesperetin exhibited an inhibitory activity on the collagen-induced platelet aggregation while they do not inhibit any of ADP-, epinephrine- and ristocetin-induced platelet aggregation. Naringin, naringenin, hesperidin and hesperetin inhibited the collagen-induced platelet aggregation by 59 to 85 %, 30 to 87 %, 59 to 93 %, and 15 to 93 %, respectively. The result shows that the flavonoid derived from citrus peels selectively inhibit the collagen-induced platelet aggregation.
Example 2: Toxicity of Orally Administered Flavonoid Derived From Citrus Peels
7 to 8 week-old, specific pathogen-free ICR female mice(8 heads) each weighing about 25 to 29 g and male mice(8 heads) each weighing about 34 to 38 g were bred under the condition of temperature 22±1°C, moisture 55±5% and photoperiod 12L/12D. Fodder(Cheiljedang Co., mouse and rat fodder) and water were sterilized and fed to the mice.
A flavonoid derived from citrus peel, naringin or hesperidin, was dissolved in 0.5% Tween 80 to a concentration of 100 mg/ml, and the solution was orally administered to the mice in an amount of 0.2 ml per 20 g of mouse body weight. The solution was administered once and the mice were observed for 10 days for signs of adverse effects or death according to the following schedule: 1, 4, 8, and 12 hours after the administration and, every 12 hours thereafter. The weight changes of the mice were recorded every day to examine the effect of the flavonoid derived from citrus peels. Further, on the 10th day, the mice were sacrificed and the internal organs were visually examined.
All the mice were alive at day 10 and, the flavonoid derived from citrus peels showed no toxicity at a dose of 1,000 mg/kg. The autopsy revealed that the mice did not develop any pathological abnormality, and no weight loss was observed during the 10 day test period. Conclusively, the flavonoid derived citrus peels is not toxic when orally administered to an animal.
Example 3: Pharmaceutical Formulation Containing Flavonoid Derived From Citrus Peels
Hard gelatin capsules were prepared using the following ingredients:
Quantity (mg/capsule)
Active ingredient (Naringin, naringenin, hesperidin, or hesperetin) 200
Vitamin C 100
Starch, dried 180
Magnesium stearate 20
Total 500 mg
Example 4: Foods Containing Flavonoid Derived From Citrus Peels
Foods containing a flavonoid derived from citrus peels were prepared as follows.
  • (1) Preparation of tomato ketchup and sauce Naringin, naringenin, hesperidin or hesperetin was added to a tomato ketchup or sauce in an amount ranging from 0.01 to 5 wt% to obtain a health-improving tomato ketchup or sauce.
  • (2) Preparation of wheat flour foods Naringin, naringenin, hesperidin or hesperetin was added to a wheat flour in an amount ranging from 0.01 to 5 wt% and breads, cakes, cookies, crackers and noodles were prepared by using the mixture to obtain health-improving foods.
  • (3) Preparation of soups and gravies Naringin, naringenin, hesperidin or hesperetin was added to soups and gravies in an amount ranging from 0.01 to 5 wt% to obtain health-improving soups and gravies.
  • (4) Preparation of ground beef Naringin, naringenin, hesperidin or hesperetin was added to ground beef in an amount ranging from 0.01 to 5 wt% to obtain a health-improving ground beef.
  • (5) Preparation of dairy product Naringin, naringenin, hesperidin or hesperetin was added to milk in an amount ranging from 0.01 to 5 wt% and various dairy products such as butter and ice cream were prepared by using the milk.
    • However, in case of cheese preparation, naringin, naringenin, hesperidin or hesperetin was added to the coagulated milk protein; and, in case of yogurt preparation, naringin, naringenin, hesperidin or hesperetin was added to the coagulated milk protein obtained after the fermentation.
    While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.

    Claims (5)

    1. A use of a flavonoid selected from the group consisting of naringin, naringenin, hesperetin and a mixture thereof for the manufacture of a medicament inhibiting a collagen-induced platelet aggregation in a mammal.
    2. The use of claim 1, wherein the mammal is human.
    3. The use of claim 1, wherein the flavonoid is administered in the form of a pharmaceutical composition or a food composition.
    4. The use of claim 3, wherein the effective amount of the flavonoid contained in the pharmaceutical composition ranges from 0.5 to 100 mg/kg body weight/day.
    5. The use of claim 3, wherein the content of the flavonoid in the food composition ranges from 0.1 to 50% by weight.
    EP99938643A 1999-08-19 1999-08-19 Flavonoids derived from citrus peels as collagen-induced platelet aggregation inhibitor Expired - Lifetime EP1123304B1 (en)

    Applications Claiming Priority (1)

    Application Number Priority Date Filing Date Title
    PCT/KR1999/000464 WO2001014396A1 (en) 1999-08-19 1999-08-19 Flavonoids derived from citrus peels as collagen-induced platelet aggregation inhibitor

    Publications (2)

    Publication Number Publication Date
    EP1123304A1 EP1123304A1 (en) 2001-08-16
    EP1123304B1 true EP1123304B1 (en) 2003-10-08

    Family

    ID=19570985

    Family Applications (1)

    Application Number Title Priority Date Filing Date
    EP99938643A Expired - Lifetime EP1123304B1 (en) 1999-08-19 1999-08-19 Flavonoids derived from citrus peels as collagen-induced platelet aggregation inhibitor

    Country Status (6)

    Country Link
    EP (1) EP1123304B1 (en)
    JP (1) JP2003507486A (en)
    CN (1) CN1322209A (en)
    CA (1) CA2345618A1 (en)
    DE (1) DE69911987T2 (en)
    WO (1) WO2001014396A1 (en)

    Families Citing this family (8)

    * Cited by examiner, † Cited by third party
    Publication number Priority date Publication date Assignee Title
    JP2005272456A (en) * 2004-02-24 2005-10-06 Takara Shuzo Co Ltd Hypoglycemic agent or cathartic agent
    NZ532666A (en) * 2004-04-30 2008-03-28 Ind Res Ltd A method of promoting angiogenesis, or treating a disease or disorder where it is desirable to promote angiogenesis, comprising administering a therapeutically effective amount of an angiogenesis promoting extract of the skin of a citrus fruit to a non-human animal
    KR100702567B1 (en) * 2004-06-09 2007-04-02 퓨리메드 주식회사 Poncirus trifoliata extract for recovering after myocardial infraction shock and pharmaceutical composition and health food containing the same
    EP2368442B1 (en) 2005-07-27 2014-12-17 Symrise AG Use of hesperetin for enhancing the sweet taste
    JP2008297279A (en) * 2007-06-01 2008-12-11 Kao Corp Agent for improving vascular endothelial function
    JP2009234924A (en) * 2008-03-25 2009-10-15 Kinji Ishida Immunity restoration agent and feedstuff composition containing the same
    JP2009234926A (en) * 2008-03-25 2009-10-15 Kinji Ishida Adrenal gland hypertrophy inhibitor and feedstuff composition or therapeutic composition containing the same
    JP6912763B2 (en) * 2014-06-27 2021-08-04 国立大学法人九州大学 Method for enhancing the functionality of catechin

    Family Cites Families (3)

    * Cited by examiner, † Cited by third party
    Publication number Priority date Publication date Assignee Title
    US4842859A (en) * 1986-09-08 1989-06-27 Yaguang Liu Pharmaceutical compositions for reducing hyperlipidemia and platelet-aggregation
    DE59309337D1 (en) * 1992-07-03 1999-03-11 Alfatec Pharma Gmbh Solid and liquid solutions of flavonoids
    JP4295840B2 (en) * 1997-12-09 2009-07-15 株式会社林原生物化学研究所 Blood circulation improving agent

    Also Published As

    Publication number Publication date
    CA2345618A1 (en) 2001-03-01
    DE69911987T2 (en) 2004-07-29
    DE69911987D1 (en) 2003-11-13
    EP1123304A1 (en) 2001-08-16
    CN1322209A (en) 2001-11-14
    WO2001014396A1 (en) 2001-03-01
    JP2003507486A (en) 2003-02-25

    Similar Documents

    Publication Publication Date Title
    US6221357B1 (en) Flavonoids derived from citrus peels as collagen-induced platelet aggregation inhibitor
    US6096364A (en) Method for lowering blood glucose level by the administration of bioflavonoid
    US6133241A (en) Bioflavonoids as plasma high density lipoprotein level increasing agent
    US5763414A (en) Hesperidin and hesperetin as 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitor
    CA2307553C (en) Citrus peel extract as inhibitor of acyl coa-cholesterol-o-acyltransferase, inhibitor of macrophage-lipid complex accumulation on the arterial wall and preventive or treating agent for hepatic diseases
    US6165984A (en) Naringin and naringenin as inhibitors of acyl CoA-cholesterol-o-acyltransferase
    US20010014669A1 (en) Method for preventing or treating elevated blood lipid level-related diseases by administering rutin and quercetin
    EP1123304B1 (en) Flavonoids derived from citrus peels as collagen-induced platelet aggregation inhibitor
    KR20070108292A (en) A food or beverage comprising agents for prevention or alleviation of hypertention and related symptoms and a process for production thereof
    CA2307890A1 (en) Hesperidin and hesperetin as inhibitor of acyl coa-cholesterol-o-acyltransferase, inhibitor of macrophage-lipid complex accumulation on the arterial wall and preventive or treating agent for hepatic diseases
    US20010002264A1 (en) Citrus peel extract as inhibitor of fatty streak formation on the arterial wall
    KR101715152B1 (en) Compound having acyl coa:cholesterol acyltransferase inhibitory and composition for prevention or treatment of cardiovascular disease comprising thereof
    JP2009185062A (en) Neovascularization inhibitor, cell proliferation inhibitor, lumen formation inhibitor, fgf inhibitor, and food or food additive, containing tocotrienol as active ingredient
    KR100291144B1 (en) Composition for preventing and treating atherosclerosis and hyperlipidemia, comprising diosmin
    JP2008120716A (en) Antiinflammatory agent
    JP4447198B2 (en) Angiogenesis inhibitor, cell growth inhibitor, tube formation inhibitor, FGF inhibitor and food or food additive containing tocotrienol as an active ingredient
    KR100291141B1 (en) Composition for preventing and treating atherosclerosis and hyperlipidemia, comprising diosmin
    EP1123096A1 (en) Bioflavonoids as plasma high density lipoprotein level increasing agent
    JPH0984568A (en) Alcohol dehydrogenase inhibitor, antibacterial agent, production of food and food
    KR100363180B1 (en) Composition for increasing plasma high density lipoprotein level, comprising bioflavonoids
    KR19990039368A (en) Blood pressure lowering composition containing hesperidin
    KR20010015494A (en) Bioflavonoid-containing composition for lowering the glocose level in blood
    JPH0653662B2 (en) Stroke preventive agent

    Legal Events

    Date Code Title Description
    PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

    Free format text: ORIGINAL CODE: 0009012

    17P Request for examination filed

    Effective date: 20010330

    AK Designated contracting states

    Kind code of ref document: A1

    Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

    17Q First examination report despatched

    Effective date: 20020806

    GRAH Despatch of communication of intention to grant a patent

    Free format text: ORIGINAL CODE: EPIDOS IGRA

    GRAS Grant fee paid

    Free format text: ORIGINAL CODE: EPIDOSNIGR3

    GRAA (expected) grant

    Free format text: ORIGINAL CODE: 0009210

    AK Designated contracting states

    Kind code of ref document: B1

    Designated state(s): DE FR GB IT

    REG Reference to a national code

    Ref country code: GB

    Ref legal event code: FG4D

    REG Reference to a national code

    Ref country code: IE

    Ref legal event code: FG4D

    REF Corresponds to:

    Ref document number: 69911987

    Country of ref document: DE

    Date of ref document: 20031113

    Kind code of ref document: P

    ET Fr: translation filed
    PLBE No opposition filed within time limit

    Free format text: ORIGINAL CODE: 0009261

    STAA Information on the status of an ep patent application or granted ep patent

    Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

    PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

    Ref country code: GB

    Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

    Effective date: 20040819

    26N No opposition filed

    Effective date: 20040709

    PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

    Ref country code: DE

    Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

    Effective date: 20050301

    GBPC Gb: european patent ceased through non-payment of renewal fee

    Effective date: 20040819

    PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

    Ref country code: FR

    Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

    Effective date: 20050429

    REG Reference to a national code

    Ref country code: IE

    Ref legal event code: MM4A

    REG Reference to a national code

    Ref country code: FR

    Ref legal event code: ST

    PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

    Ref country code: IT

    Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

    Effective date: 20050819